4微生物培养基Culturalmedium

4.微生物培育基CulturalmediaAculturalmediumisthesubstanceinwhichaspecificorganismlivesandgrows.Itmustcontaintheessentialnutrientsneededforthemicrobetogrow.Agoodmediumisveryimportanttothesuccessofanindustrialfermentation.Themediumsuppliesnutrientsforgrowth,energy,buildingofcellsubstance,andbiosynthesisoffermentationproducts.4.1培育基的类型及功能

Classificationsofmediaandtheirfunctions按纯度分类：--合成培育基chemicallydefinedmedia:compoundidentityandconcentrationofallcomponentsareknown.Syntheticmedia--天然培育基chemicallyundefinedmedia:ingredientsincludeoneormorechemicallycomplexsubstancessuchasproteinhydrolysatesandextracts.CrudemediaThechoiceofdefinedorundefinedmediumisdependentuponitsapplication.---Chemicallydefinedmediaareusefulinbiochemicalormetabolicstudiesoforganisms.--Generallaboratorygrowthmediaandindustrialmediaareoftenchemicallycomplex.Suchas花生饼、蛋白胨等。普通不需外加微量元素(traceelements)、维生素等。

按形状分类：-Solidmedia：suitabletocultureandpreservespeciesandspores;cultivatingfungi.-Liquidmedia:80%-90%water,industrialfermentationmedia.-Semi-solidmedia:liquidmedium+agar主要用于鉴定细菌、察看细菌运动特征等。按用途分类孢子培育基、种子培育基和发酵培育基1.孢子培育基sporemedia要求：使菌体迅速生长，产生较多的优质孢子，不易引起菌种发生变异(variation)。根本配制要求：⑴notrichinnutrition,especiallyorganicnitrogensoure.⑵optimalmineralsaltconcentration⑶optimalpHandhumidity消费上常用孢子培育基：麸皮、大米、小米培育基；由葡萄糖、蛋白胨、牛肉膏和食盐等配制的琼脂斜面培育基。大米和小米常用作霉菌孢子培育基，因它们含氮少、疏松、外表积大，是较好的孢子培育基。水分控制在21％－25％。种子培育基inoculummedia:种子培育基是供孢子发芽、生长和大量繁衍菌丝体，并使菌丝体长得粗壮，成为活力强的‘种子’。Inoculummediaareusuallylessnutritiousthanareproductionmedia,andtheyusuallycontainaconsiderablylowerlevelofthemainnutritivecarbonsource.普通种子培育基常包括有机氮源和无机氮源最后一级的种子培育基的成分最好接近发酵培育基。发酵培育基productionmedia发酵培育基是供菌体生长、繁衍和合成产物之用。要求：既要使种子接种后能迅速生长，到达一定的菌丝浓度，又要使长好的菌体迅速合成所需产物。配制要求：其组成除有菌体生长所必需的元素和化合物外，还需有合成产物所需的特定元素、前体和促进剂等。Inadditiontothecarbonandnitrogencompounds,amediumcontainsinorganicsalts,vitamins,growthfactors,precursorsoffermentationproducts.Microbialgrowthunderindustrialfermentationconditionsusuallyutilizesa“luxurymetabolism〞oftheorganisms.Thus,goodyieldsoffermentationproductsoccuronlyifrelativelylargeamountsofcarbon,andpossiblynitrogen,arechanneledthroughthemetabolicpathwaysofthemicroorganism.4.2发酵培育基的成分及来源

CompositionofmediaandtheirsourcesTheparticularcompositionofafermentationmediumcanbesimpletocomplexdependingontheparticularmicroorganismanditsfermentation.Autotrophic(自养的)microorganismsrequireonlythesimplestofinor-ganicmedia.Heterotrophic(异养的)microorganismslacktheabilitytosynthesizemanyoftheirsustenanceandgrowthrequirements.4.2.1碳源carbonsourcesCarbonsourceisoneofmaincompositionofmedia.Thefunctionsare:providingcarbonsandenergy;thecomponentsforsynthesisofproducts.Thesecanincludesimplesugars,complexcarbohydrates,alcohols,aminoandotherorganicacids,andshort-chainlipids.常用的碳源有糖类、油脂、有机酸和低碳醇。蛋白质、氨基酸也可用作碳源。4.2.1.1糖类sugarsSugarsusedmainly:glucose,molasses(糖蜜),dextrin(糊精).Glucose:mostmicrobescanutilizeit.速效碳源、highconcentrationspeedrespirationupDOnoenoughintermediates(中间产物〕accumulationinhibitinggrowthmolasses(糖蜜)Beet(甜菜)andcanemolassesareby-productsofthesugarindustry.containing50%-75%fermentablesugars,mainlysucrose.Alsocontainingnitrogenouscompounds,vitaminsandminerals.CheaperStarchanddextrin(糊精)Starch:----widelyusedinfermentationindustry---starchhydrolyzeusingmicrobialamylasedextringlucose-Mostcommonusedstarches:corn,wheat,potatostarches.Alsousingcornflour.--advantages:缓效碳源、cheaperthanglucose油和脂肪oilsandfatsOilandfatsarealsousedascarbonsourcesbecausesomemicrobescontainlipase(脂肪酶)Fatglycerol(甘油)andfattyacids

oxidizeCO2+water,releasingmuchenergyProvidingenoughO2isimportant.常用的油：豆油、菜子油、猪油、鱼油、棉子油等。有机酸organicacidsSomemicrobescanuseorganicacidsascarbonsources,suchaslacticacid,citricacid,aceticacid.pHofculturalmediumwillincrease.CH3COONa+O22CO2+H2O+NaOH烃和醇类hydrocarbonandethanol石油及裂解产物如正烷烃也可用作carbonsources.自然界中能同化乙醇的微生物和能同化糖质的微生物一样普遍。4.2.2氮源nitrogensources氮源主要用于构成细胞物质〔氨基酸、蛋白质、核酸等〕和含氮代谢物。有机氮源organicnitrogensources常用的:花生饼、棉子饼、玉米浆、玉米蛋白粉、蛋白胨、酵母粉、鱼粉、蚕蛹粉、尿素、废菌丝体和酒糟等。organicnitrogensourcesmicrobialproteinaseaminoacidsmetabolizedbymicrobes.organicnitrogensourcescontainrichproteins,peptides,aminoacids,vitaminsandgrowthfactors.在含有机碳源的培育基中，微生物常表现出生长旺盛、菌丝或细胞浓度增长迅速的特点。有些微生物对氨基酸有特殊的需求。如缬氨酸可提高红霉素的发酵单位。玉米浆CornsteepLiquorCornsteepliquoristhewaterextractby-productresultingfromthesteeping(浸泡)ofcornduringthecommercialproductionofcornstarchandothercornproducts.Ofthe50%solidsofcornsteepliquor,nearlyhalfislacticacid.Therestincludesaminoacids,glucoseandotherreducingsugars,salts,vitamins,andprecursors(前体)suchasthoseforthepenicillinmolecule.Thehighlacticacidcontentofcornsteepliquorresultsfromthegrowthoflacticacidbacteriaduringitsmanufacture.Thisvariationincomposition,attimes,canleadtopoorreproducibilityofanindustrialfermentation.尿素ureaUreaisalsoacommonlyusednitrogensource.Itsfeature:---singlecomponent,nonutritionalfeatureofcomplexnitrogensources;---cheaper,oftenusedinproductionofpenicillinandglutamicacid.yeastextractpeptones有机氮源除了作为菌体生长繁衍的营养外，有的还是产物的前体。缬氨酸、半胱氨酸和-是合成青霉素和头孢菌素的主要前体；甘氨酸可作为L-丝氨酸的前体等。无机氮源inorganicnitrogensource常用的：氨水(ammonia)、铵盐(ammonium)和硝酸盐(nitrate)Microbescanutilizeitfaster.Afteritisutilized,thepHofmediumwillbechanged.(NH4)2SO42NH3+H2SO4NaNO3+4H2NH3+2H2O+NaOH生理酸性物质：NH4酸性物，硫酸铵生理碱性物质：NH4碱性物，硝酸铵正确运用生理酸碱性物质Exceptusingasanitrogensource,ammoniaisoftenusedtoadjustpHofmedia.Stirringisnecessary.4.2.3无机盐及微量元素

mineralsaltsandtraceelementsMineralsaltsandtraceelementsareneededwhenmicrobialcellsgrowandreproduce.Mostcommonmineralsaltsandtraceelements:磷(phosphorus)、镁(magnesium)、硫(sulphur)、钾(potassium)、钠(sodium)、铁(iron)、氯(chlorine)、锰(manganese)、锌(zinc)、钴(cobalt)、钙等作为微生物细胞生理活性物质组成或生理活性作用的调理物。Whentheirconcentrationislow,theyshowastimulationfunctionforcellgrowandproductsynthesis.Whentheirconcentrationishigh,theywillinhibitthecellgrowth.Theiroptimalconcentrationsdependonthespeciesandstrains.无机盐成分普通所用的浓度范围成分浓度g/L成分浓度g/LKH2PO4MgSO4·7H2OKClCaCO3FeSO4·4H2O1.0~4.00.25~3.00.5~12.05~170.01~0.1ZnSO4·8H2OMnSO4·H2OCuSO4·5H2ONa2MoO4·2H2O0.1

~1.00.01~0.10.003~0.010.01~0.1Theseelementsareaddedintoculturalmediaintheformoftheirsalts.Exceptsyntheticmedia,cobalt钴,copper,iron,manganese锰,zinc,molybdenum钼arenotaddedintocrudemedia.Phosphoruselementisoneofnucleicacidsandproteinscomposition,onecomponentofATP,energytransfer.Phosphoruscanstimulatemicrobialgrowthandmetabolism.Butitinhibitscellgrowthwhenitsconcentrationishigh.许多次级代谢过程对磷酸盐浓度的接受限制比生长繁衍过程低，故必需严厉控制。Magnesiumdoesnotinvolvethecellinstitution.ButMgistheactivatorformanyimportantenzymes.如己糖磷酸化酶、柠檬酸脱氢酶等镁离子可提高抗生素产生菌的抗生素耐受才干，如链霉素、卡那霉素等产生菌。常以硫酸镁的方式参与培育基。硫存在于细胞的蛋白质中，是含硫氨基酸的组分和某些辅酶的活性基，如辅酶A(coenzymeA),谷胱甘肽等。硫是某些产物如青霉素、头孢菌素等分子的组成部分，在培育基中参与Na2SO4等含硫化合物作硫源。铁(iron)是细胞色素、细胞色素氧化酶和过氧化氢酶的成分，因此铁是菌体有氧氧化必不可少的元素。Iniron-madefermentor,generallyironionconcentrationcanbe30µg/ml.Soironcontainingcompoundsarenotnecessaryforindustrialmedia.Chlorinedoesnothavenutritionalfunctionsformostmicroorganisms,butitisnecessaryforcellgrowthofhalophile.一些产含氯代谢物如金霉素和灰黄霉素等的发酵中，除天然含有外，通常还需参与0.1%氯化钾。在啤酒消费中，20－60mg/ml的氯对酶和酵母有一定的促进作用。Sodium,potassium,calciumarenotcomponentsofcellconstitution,theyarestillnecessarycompositionforculturalmedia.钠、钾离子与维持细胞的浸透压有关。钾离子是许多酶的激活剂，能促进糖代谢。钙是某些酶(如蛋白酶)的激活剂，还参与细胞膜通透性的调理。培育基中钙盐过多时，会构成磷酸钙沉淀。。锌、镁、钴、锰等是某些酶的辅基或激活剂。镁离子还可提高抗生素消费菌对本人所产生抗生素的耐性；钴既是一些酶的激活剂，又是VB12的组成元素，发酵中参与一定量的钴盐，能使VB12的产量提高数倍；锰对于羧化作用是必需的，糖代谢中许多酶的活性都与锰有关。4.2.4WaterMaincompositionofmediaFunctions:---joindirectlysomemetabolisms---solvents---providingaphysiologicalenvironmentsforcellgrowthandsynthesisofproducts.Effectofwaterqualityonfermentation4.2.5生长因子、前体、产物促进剂(1)生长因子growthfactorsDefinition:smallamountoforganiccompoundsnecessaryformicrobialgrowth.Features:nosynthesisbyitself.Examples：aminoacids,purines,vitaminsSources:normallyorganicnitrogensource.Cornsteepliquor.(2)前体precursorsDefinition:substancesaddedpriortoorsimultaneouslywiththefermentation,whichareincorporatedwithoutanymajorchangeintothemoleculeofthefermentationproduct,andwhichgenerallyservetoincreasetheyieldorimprovethequalityoftheproduct.前体是指某些化合物参与到发酵培育基中，能直接被微生物在生物合成过程中结合到产物分子中去，而其本身的构造并没有多大变化，但是产物的产量却因参与前体而有较大的提高。Examples：苯乙酸青霉素G苯氧乙酸青霉素V发酵过程中所用的一些前体物质产品前体产品前体青霉素G青霉素V金霉素灰黄霉素红霉素苯乙酸苯氧乙酸氯化物氯化物正丙醇核黄素类胡萝卜素L-异亮氨酸L-色氨酸L-丝氨酸丙酸盐β-紫罗酮

-氨基丁酸邻氨基苯甲酸甘氨酸(3)产物促进剂产物促进剂productstimulatorDefinition:substancesareneithernutrientsnorprecursors,andincreaseproductyieldafteraddingthem.Examples:Tween(0.1%)amylase(淀粉酶),cellulase(纤维素酶)大豆酒精提取物cellulaseMechanisms:notveryclearPossiblereasons:---enzymeinducers诱导剂---improvementofcellpermeability浸透性---protectiononenzymeactivity4.3培育基的设计及优化

mediadesignanditsoptimization

ImportanceofmediadesignCurrentsituationsofmediadesign:manyfactorsempirical阅历+scientific4.3.1培育基成分选择的原那么(1)菌体的同化才干：largemolecules+enzymessmallmoleculesSelectionofcarbonsources:

glucose,starches不同糖化工艺所得糖液质量的比较项目酸法酸酶法双酶法葡萄糖值(DE值)葡萄糖含量％灰分％色度羟甲基糠氨％葡萄糖收率91861.610.00.3095930.40.30.008较酸法高5％98970.10.20.003较酸法高10％Selectionofnitrogensources:---Ifthemicrobeproducesproteolyticenzymes,avarietyofcrudenitrogensourcessuchassoybeanmealcanbeused.--Ifthemicrobelackofproteases,theorganicnitrogensourceshouldbehydrolyzed.豆饼粉＋water+hydrochloricacid盐酸,pH1.0100°Caminoacids.(2)代谢物的阻遏和诱导---葡萄糖效应---combinationoforganicandinorganicnitrogen/carbonsources---enzymeagentproduction:Formanyinducedenzymes,starchanddextrinarecommoncarbonsource.

碳源对生长和产酶的影响碳源地衣芽孢杆菌细胞量g/l

淀粉酶活/(U/ml)glucose